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Bionanotechnology

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Synthetic forms created to contain manganese oxide, uranyl oxyhydroxide, iron sulfide, ... tissue engineering Nanomedicine (1 lecture) Nanotherapeutics, ... – PowerPoint PPT presentation

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Title: Bionanotechnology


1
Bionanotechnology
  • Dr Cait MacPhee (cem48_at_cam.ac.uk)
  • Dr Paul Barker (pdb30_at_cam.ac.uk)
  • Mondays 12 pm, Tuesdays 11 am

2
Syllabus
  • The molecules of life
  • Proteins (6 lectures)
  • background
  • as components in nanodevices
  • biomolecular electronic devices
  • electron transport and photosynthesis
  • as fibrous materials
  • in motion molecular motors
  • DNA (3 lectures)
  • background
  • as components in nanodevices part I
  • as components in nanodevices part II
  • Lipids (1 lecture)
  • background as components in nanostructures
    artificial cells (liposomes and membrane
    nanotubes)
  • Bio-inorganic composites (1 lecture)
  • composites including butterfly wings, diatoms,
    mineralisation
  • The whole cell
  •  Cell mechanotransduction (1 lecture)
  • bringing together physical, life, and applied
    sciences bone cell mechanobiology
  •  
  • Cell motility (1 lecture)
  • how cells travel and navigate through 2- and 3
    dimensional environments
  •  
  • Biomaterials (1 lecture)
  • surface science/ surface chemistry tissue
    engineering
  •  
  • Nanomedicine (1 lecture)
  • Nanotherapeutics, real and imagined
  •         Qdots and developmental biology
  •  
  • Ethical considerations (1 lecture)
  • risk/benefit analysis focusing on
    bio-nanotechnology

3
So why are we interested?
  • Biological systems inherently good at
    self-assembly
  • Multitude of functions evolved over millenia
  • Bacterial expression systems allow us to
    artificially evolve unnatural functions

4
Protein Function
  1. Structural collagen, elastin Force transmission
  2. Mobility actin/myosin, flagella Linear/rotary
    motors
  3. Receptors insulin receptor
  4. Ligands insulin
  5. Immune system antibodies...
  6. housekeeping chaperones... Quality control
  7. signalling kinases... Amplification
  8. Enzymes proteases Chemical/ photochemical work
  9. Storage and Transport haemoglobin

Lock and key recognition
5
How do you produce a bucket of protein?
DNA
RNA
Protein
  • Expression systems are based on the insertion of
    a gene into a host cell for its translation and
    expression into protein
  • Insert the DNA coding the protein of interest
    into a plasmid a small, circular piece of DNA
    that is found in E. coli and many other bacteria
  • generally remain separate from the bacterial
    chromosome
  • carry genes that can be expressed in the
    bacterium
  • generally replicate and are passed on to daughter
    cells along with the chromosome

6
Protein Expression
  • Results in a population of bacteria replicating
    many copies of a segment of foreign DNA
  • Many recombinant proteins can be expressed
    (produced) to high levels in E. coli systems
  • Plus
  • Unnatural amino acids
  • Artificial (accelerated) evolution

7
..but!
  • Protein sequence space is impossibly large
  • 2060 1078 gt number of particles in the universe
  • More variants can be made than can be reasonably
    tested
  • 1015 can be made in a test tube
  • can only be assayed by enrichment strategies
  • 107 can be tested with a genetic selection
  • 106 -107 can be tested with an ultra
    high-throughput screen
  • single cell assays
  • 104-105 in microtiter plates
  • Really need to be able to screen at least 1000
    samples per week for in vitro evolution

8
(Nanoscale) problems?
  • Inertia/ viscosity Thermal noise Gravity
    predicting sequence-structure-function
    relationship
  • Water
  • The hydrophobic effect
  • Depletion forces
  • Entropic effect
  • Large objects (i.e. proteins) are surrounded by a
    depletion zone of thickness equal to the radius
    of small particles (i.e. solutes)
  • Elimination of the depletion zone increases the
    entropy, thereby decreasing the free energy of
    the solute
  • Short range (2R) interaction

9
Some examples
  • Viral toolkit
  • S-layers
  • HSP60
  • Ferritin
  • Bacteriorhodopsin

10
Viruses
  • A bacteriophage is a virus capable of infecting
    and reproducing in bacteria
  • Filamentous phage are comprised of a
    single-stranded DNA molecule encased in a protein
    cylinder
  • Protein cylinder is assembled from five coat
    proteins
  • Foreign DNA can be incorporated into the genome
    and displayed as a fusion to a coat protein

11
M13 phage
  • Dimensions
  • 6.5nm in diameter
  • Length dependent on genome but wild type
    approximately 930nm
  • Mass 16.3MD of which 87 is protein
  • Sophisticated life cycle
  • Infection transfers genetic material into host
    with little disruption.
  • Late infection stages perturb host membrane for
    release of virions, but M13 is non-lytic. Does
    not kill its host turns it into a virus factory.

12
Phage Peptide Protein Libraries
  • Libraries of peptide or protein sequences are
    constructed and cloned into the phage genome
  • By cloning large numbers of DNA sequences into
    the phage, display libraries are produced with a
    repertoire of many billions of unique displayed
    proteins (i.e. a peptide of 6 amino acids 64
    million variants).
  • One foreign insert per phage a single
    polypeptide sequence displayed per particle
  • All copies of g3 have the same polypeptide
    displayed (multivalency)
  • Select for desired activity/ function

13
Selecting for non-biological functions
  • Phage selected for nucleation of semiconducting
    CdS and ZnS particles

14
Single crystal nanowires
  • Mineralisation nucleation from metal salt
    solution at low temperatures to yield
    crystallographically uniform orientation of
    nanocrystals
  • Crystals cannot fuse due to presence of
    nucleating peptide
  • Anneal at 350C
  • Successful for ZnS and CdS single crystal
    nanowires
  • Ferromagnetic CoPt and FePt systems selected for
    development of low-dimensional magnetic materials

Mao CB, Solis DJ, Reiss BD, et al. Science 303
213-217 2004
15
S-layers
  • Cover the cell surfaces of a wide range of
    bacteria
  • Hypothesized to be involved in protection,
    molecular sieving, ion trapping, cell adhesion,
    surface recognition, and morphogenesis.
  • Self-assemble into 2-D crystalline lattices in
    vitro, on surfaces, polymers, and liquid-air
    interface
  • Lattices 5-10 nm thick and containing pores of
    2-8 nm (depending on the species).

16
S-layers
  • S-layers on solid supports nucleation and growth
    of CdS and Au nanoparticles in pores.
  • Defined size (5 nm), defined superlattice
    symmetry (13 nm spacing)
  • Preformed citrate-modified gold nanoparticles
    self-assemble in a hexagonal array (7000
    particles/ mm2, separation 10.4 nm)
  • Hard disk drives currently store information at
    70 Gbit/in2 density restricted by coarse
    patterning/ coarse grain size and
    superparamagnetic effect
  • S-layers potentially 4 Tbit/ in2

Bergkvist, M., Mark, SS., Yang X. et al., J Phys
Chem B 108, 8241-8248 2004
17
S-layers
  • S-layer acts as mask to generate surface pattern
    in Si via plasma etching.
  • 10 nm diameter magnetic dots, separation 22 nm
  • No superparamagnetic effect
  • System is ferromagnetic at room temperature, with
    magnetization in the plane of the sample - system
    lies between a continuous film and an array of
    independent dots

S-layer on Si
Cr (e-beam)
Plasma etch
Fill Pd/ Fe
Remove mask
Malkinski L, Camley RE, Celinski Z, et al. J Appl
Phys 93 7325-7327, 2003
18
S-layers
  • S-layer self-assembles on Si wafer
  • Bring into direct contact with a photomask and
    expose to deep UV irradiation (ArF, 193 nm _at_
    200 mJ cm-2).
  • S-layer entirely ablated from Si substrate to
    generate long-range crystalline pattern
  • A natural resist with excellent (5 nm) edge
    resolution?

Pum D, Sleytr UB Trends Biotechnol 17 8-12 1999
19
HSP60
  • In nature, chaperonins are ubiquitous and
    essential subcellular structures responsible for
    ensuring proteins are correctly folded in the
    cell
  • Composed of 14, 16 or 18 subunits called heat
    shock proteins arranged as two stacked rings.
  • The extremophile S.shibatae grows at 85C at pH
    2. Chaperonin is octadecameric with 9 subunits
    per ring 18 nm tall ? 17 nm wide.
  • Thermostable, of known sequence, and can adopt
    higher-order structures such as 2-D crystals

McMillan RA, Paavola CD, Howard J, et al. Nat
Mater 1 247-252, 2002
20
HSP60
  • Mutant protein assembled into hexagonally packed
    2-D arrays 20 mm diameter.
  • Added commercial gold QDs
  • Mutant protein with 3 nm thiol ring bound and
    ordered 5 nm particles in pore region did not
    bind/ order 10 nm 15 nm particles
  • Mutant protein with 9 nm thiol ring bound and
    ordered 10 nm particles in pore region.
  • Centre-to-centre spacing of 16 nm edge to edge
    spacing 6-10 nm
  • Also bound ordered 4.5 nm Cd/Se-ZnS QDs -
    mottled

21
HSP60
  • React gold nanoparticles (1.4 nm) with protein
    subunits prior to self-assembly
  • Observed ordered hexagonally spaced inclusions,
    with density 9 times greater than single gold
    nanodot
  • Defect tolerant?
  • Protein intrinsic to design, but super-stable
    complex

22
Ferritin
  • 24 subunits, 8-9 nm central cavity
  • Naturally chelates iron (III) as ferrihydrite
  • A set of moulds for making nanometer-scale
    machine parts with controlled shapes and
    compositions?
  • Synthetic forms created to contain manganese
    oxide, uranyl oxyhydroxide, iron sulfide, CdS,
    Ni, Co/Pt
  • Co/Pt and Fe-Pt selected as thermally stable at
    3-4 nm diameter

23
Ferritin in magnetic media?
  • Superparamagnetic Co-Pt prepared within
    apoferritin in solution
  • Self-assembles at an air/water interface giving a
    well-ordered array of L-ferritin on Si.
  • Anneal at 500-650ºC for 60 min to form
    ferromagnetic L10 phase and carbonise protein
    (UV/ ozone treatment decreases sintering)
  • Hard disk drives information density restricted
    by coarse patterning/ coarse grain size and
    superparamagnetic effect
  • Self-assembly of ferritin followed by
    carbonisation maximum 5,000-10,000 Gbits in-2

24
Ferritin organised on S-layers
50nm
25
Bacteriorhodopsin
  • Found mainly in the halophilic archae
    halobacteria
  • Exist in a 2D crystalline form in the membrane
    (purple membrane)
  • Stable in 5 M (25 ) NaCl, exposure to sunlight
    in oxygen (years), temperatures 140ºC when dry,
    pH values 0-12, enzyme digestion
  • Converts light energy into chemical energy
  • Simplest photosynthetic machinery drives an ATP
    synthetase in the membrane under anaerobic
    conditions

26
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27
Bacteriorhodopsin
28
Bacteriorhodopsin
  • Photon absorbed photochemical switch in the
    retinal (isomerisation) driving proton pump,
    proton motive force drives ATP synthase
  • Thermal relaxations regenerate ground state.
  • Non physiological conditions and intense light
    can access branched photocycle and populate the
    P/Q forms
  • P/Q are stable for decades do not undergo
    thermal relaxation

29
BR in holographic memory
30
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31
BR in holographic memory
Advantages? Low cost of molecules Self
assembly at some stages. No moving parts Storage
density? 1x1x2in 1Tbyte Limitations? protein
purity Laser focusing
32
Summary
  • Proteins have useful self-assembly properties
    (Viral toolkit, S-layers, HSP60, Ferritin,
    Bacteriorhodopsin)
  • Genetic engineering allows production of entirely
    new functional sequences
  • Protein not necessarily intrinsic to design, but
    can select for (or evolve) very stable varieties

33
Syllabus
  • The molecules of life
  • Proteins (6 lectures)
  • background
  • as components in nanodevices
  • biomolecular electronic devices
  • electron transport and photosynthesis
  • as fibrous materials
  • in motion molecular motors
  • DNA (3 lectures)
  • background
  • as components in nanodevices part I
  • as components in nanodevices part II
  • Lipids (1 lecture)
  • background as components in nanostructures
    artificial cells (liposomes and membrane
    nanotubes)
  • Bio-inorganic composites (1 lecture)
  • composites including butterfly wings, diatoms,
    mineralisation
  • The whole cell
  •  Cell mechanotransduction (1 lecture)
  • bringing together physical, life, and applied
    sciences bone cell mechanobiology
  •  
  • Cell motility (1 lecture)
  • how cells travel and navigate through 2- and 3
    dimensional environments
  •  
  • Biomaterials (1 lecture)
  • surface science/ surface chemistry tissue
    engineering
  •  
  • Nanomedicine (1 lecture)
  • Nanotherapeutics, real and imagined
  •         Qdots and developmental biology
  •  
  • Ethical considerations (1 lecture)
  • risk/benefit analysis focusing on
    bio-nanotechnology
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